Therapeutic target for inflammatory bowel disease and application thereof

Abstract

The application discloses a therapeutic target of inflammatory bowel disease and application thereof. The application researches and finds that NO can be combined with the enzyme active site of CYP450 family members, thereby inhibiting the activity, finally inhibiting the production of pathogenic factors GM-CSF, and relieving enteritis. Based on this, an inflammatory bowel disease product, a kit and an analysis system are developed. The application can provide a new strategy for preventing, detecting and treating intestinal inflammatory diseases, and effectively improve intestinal homeostasis imbalance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, especially relates to the technical field of drugs for treating intestinal inflammatory diseases and analytical detection, and specifically relates to a therapeutic target for inflammatory bowel disease and application thereof. BACKGROUND

[0002] Intestinal immune regulation is crucial for maintaining intestinal homeostasis. The intestinal immune system needs to achieve a delicate balance between resisting pathogen invasion and tolerating symbiotic bacteria and dietary antigens. Once this balance is broken, it will lead to intestinal inflammatory diseases, such as inflammatory bowel disease (IBD). IBD includes ulcerative colitis (UC) and Crohn's disease (CD), which is a chronic and recurrent intestinal inflammatory disease with a high incidence in industrialized countries. In recent years, with the advancement of industrialization in China, the incidence of IBD in China has also shown a rapid growth trend.

[0003] The pathogenesis of IBD involves complex interactions of multiple factors, including genetic mutations, environmental factors and immune regulation abnormalities. Among them, elucidating the molecular mechanisms of immune regulation abnormalities in IBD patients is crucial for developing effective treatment strategies. However, current treatment methods targeting immune regulation, such as blocking tumor necrosis factor (TNF), interleukin (IL)-12 and IL-23, have not achieved ideal efficacy, which indicates that new pathogenic mechanisms need to be further explored to reveal markers for sensitive populations.

[0004] In recent years, a large number of studies have confirmed that the activation and function of immune cells are precisely regulated by metabolic networks. Among the large number of metabolic products produced by the host and intestinal flora metabolism of dietary components, biologically active gases represented by nitric oxide, hydrogen sulfide and carbon monoxide have attracted attention because they can participate in various physiological processes including immune response. In particular, nitric oxide (NO) has been confirmed to be related to the pathogenesis of IBD, but its specific mechanism in IBD is still unclear. SUMMARY

[0005] The first object of the present application is to provide a therapeutic product for inflammatory bowel disease. The second object of the present application is to provide a detection reagent and kit for nitric oxide (NO) deficiency related inflammatory bowel disease and its risk of disease. The third object of the present application is to provide an analysis system for detecting inflammatory bowel disease caused by nitric oxide (NO) deficiency.

[0006] Anti-CD40-induced enteritis model is a common mouse model for studying intestinal inflammation caused by innate immunity. In this model, ILC3 produces the cytokine GM-CSF (from the geneCSF2 The coding (encoding) is the main pathogenic factor, which can recruit neutrophils and pro-inflammatory macrophages, thereby leading to tissue damage.

[0007] During the research process of this invention, it was discovered that:

[0008] ILC3 cells express members of the CYP450 family (mouse cells express Cyp4f13, etc., and humans express CYP4F3, etc.). These P450 metabolic enzymes can metabolize polyunsaturated fatty acids (represented by arachidonic acid, AA) and produce ROS. ROS further promotes NF-κB activity, thereby promoting... CSF2 The expression of iNOS (genotype 1) is observed in tissues during enteritis. NOS2 (Encoding), synthesizing NO. NO can bind to the active sites of enzymes in the CYP450 family, thereby inhibiting their activity and ultimately suppressing the production of the pathogenic factor GM-CSF, alleviating enteritis. When NOS2 In cases of deficiency, enteritis in mice (anti-CD40-induced enteritis model) is exacerbated. P450 inhibitors can also (replacing the role of NO) bind to the enzyme's active site, thereby inhibiting GM-CSF production and alleviating enteritis.

[0009] Analysis of human tissue samples revealed that human ILC3 can bind NO and generate ROS using arachidonic acid (AA). Treatment with the P450 inhibitor HET0016 inhibited the ability of human ILC3 to generate ROS and GM-CSF. iNOS expression varied considerably in the intestinal tissues of patients, but was significantly higher overall than in healthy controls. In patient tissues, the level of NO binding by ILC3 was inversely proportional to the activity of enteritis.

[0010] Based on the above findings

[0011] The first objective of this invention is achieved through the following technical solution:

[0012] Applications of CYP450 members that can bind to NO as targets in the development or design of products with at least one of the following functions (1)-(3):

[0013] (1) Treatment and / or prevention of inflammatory bowel disease;

[0014] (2) Detection of inflammatory bowel disease;

[0015] (3) Inhibit the production of inflammatory factors.

[0016] The products are pharmaceuticals, diagnostic reagents, diagnostic systems, and kits. Among the CYP450 molecules, CYP4F3 is a member capable of binding NO.

[0017] Use of a substance inhibiting the enzyme activity or expression of a member of CYP450 capable of binding NO in the preparation of a medicament for treating and / or preventing inflammatory bowel disease.

[0018] Further, the substance inhibiting the enzyme activity of a member of CYP450 capable of binding NO includes N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016) and its derivatives, ABT-1, Verapamil hydrochloride, ZINC05626394, Ketoconazole and Abiraterone.

[0019] Further, the substance inhibiting the enzyme activity of a member of CYP450 capable of binding NO includes N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016) and its derivatives, ABT-1, Verapamil hydrochloride, ZINC05626394, Ketoconazole and Abiraterone.

[0020] Further, the medicament is an oral medicament, an enema medicament or an injection medicament; when the medicament is an oral medicament, it is a powder, a tablet or an oral liquid; when the oral powder or tablet is used, the content of HET0016 is not less than 2%, and when the oral liquid is used, the content of HET0016 in the solute is not less than 5%; when the medicament is an enema medicament, the content of HET0016 in the solute is not less than 2%; and when the medicament is an injection medicament, the content of HET0016 in the solute is not less than 5%.

[0021] Further, the medicament is a combination medicament, and the components of the combination medicament further include one or more of 5-aminosalicylic acid, a glucocorticoid, an immunomodulatory drug, a biological agent and an oral small molecule reagent, wherein the biological agent includes an anti-cytokine drug.

[0022] The second object of the present application is achieved by the following technical solution:

[0023] A kit for detecting inflammatory bowel disease caused by NO deficiency, comprising a quantitative reagent for detecting the expression amount of NO deficiency related protein; the quantitative reagent for detecting the expression amount of NO deficiency related protein includes a quantitative reagent for detecting NO synthase, a quantitative reagent for detecting GM-CSF and a quantitative reagent for detecting a member of CYP450 capable of binding NO.

[0024] The NO synthase includes any one or more of NOS, iNOS and eNOS, and the member of CYP450 capable of binding NO includes CYP4F3.

[0025] In a further optimized solution, the NO synthase is iNOS.

[0026] The quantitative reagent for detecting iNOS includes the gene of iNOS. NOS2Primer: nucleotide sequence as shown in SEQ ID NO. 1 and SEQ ID NO. 2 (For: 5'-TTCAGTATCACAACCTCAGCAAG-3', Rev: 5'-TGGACCTGCAAGTTAAAATCCC-3');

[0027] The quantitative kit for detecting GM-CSF includes its gene CSF2 Primer: nucleotide sequence as shown in SEQ ID NO. 3 and SEQ ID NO. 4 (For: 5'-TCCTGAACCTGAGTAGAGACAC-3', Rev: 5'-TGCTGCTTGTAGTGGCTGG-3');

[0028] The quantitative kit for detecting CYP4F3 includes its gene CYP4F3 Primer: nucleotide sequence as shown in SEQ ID NO. 5 and SEQ ID NO. 6 (For: 5'-CAACCCCCGAAACGGAATTG-3', Rev: 5'-GAAGATGCGGACGATTGCG-3').

[0029] The implementation of the present scheme is based on the following research:

[0030] In the present research, it is found that the expression of enzymes related to NO synthesis is elevated during human and mouse intestinal inflammation, and the over-activation of ILC3 is inhibited by the synthesis of NO.

[0031] Specifically, the RNA-seq data of intestinal tissues of IBD patients and various mouse intestinal inflammation models in the GEO database are analyzed to determine NOS2 The expression is consistently up-regulated in human and mouse intestinal inflammation. By using NOS2 Deficient mice, anti-CD40-induced intestinal inflammation modeling is performed, and its intestinal inflammation is significantly aggravated, and the ability of intestinal ILC3 to produce pathogenic cytokine GM-CSF is significantly increased.

[0032] Further single-cell RNA-seq analysis of intestinal ILC3 in anti-CD40-induced intestinal inflammation modeling is performed, and gene knockout mice are used for verification, and the target molecule CYP4F13 of nitric oxide is found. Nitric oxide limits the over-activation of mouse ILC3 by inhibiting the activity of CYP4F13, and human ILC3 expresses a protein CYP4F3 highly homologous to mouse CYP4F13. Therefore, this group of genes can be used to construct a detection reagent and kit for intestinal inflammatory diseases related to nitric oxide deficiency and the risk of suffering from the diseases.

[0033] The second kit for detecting inflammatory bowel disease caused by NO deficiency comprises quantitative reagents for detecting the intracellular NO content of intestinal immune cells, wherein the intestinal immune cells are ILC3, T cells and B cells.

[0034] Further, the flow cytometry antibodies for detecting immune cells are shown in Table 1, and the reagent for detecting the intracellular nitric oxide level is DAF-FA DA (Biyun Tian, S0019S).

[0035] Table 1: Flow cytometry antibodies for detecting intestinal immune cells

[0036] Antibody Name Target Antigen Clone Number Fluorochrome Supplier / Catalog Number CD3 T cell surface antigen OKT3 eFluor 450 BioLegend, 317313 CD19 B cell surface antigen SJ25C1 eFluor 450 BioLegend, 363035 IL-7Rα ILC surface antigen A7R34 Bv650 BioLegend, 135043 c-Kit ILC3 surface antigen ACK2 Bv785 BioLegend, 135138 CRTH2 ILC2 surface antigen BM16 PE / CF954 BioLegend, 350135 CD45 Immune cell surface antigen HI30 APC / Cy7 BioLegend, 304014

[0037] The implementation of the present scheme is based on the following research:

[0038] The present application researches and finds that ILC3 of human and mouse can bind extracellular NO, and the overactivation thereof is inhibited by extracellular NO.

[0039] Specifically, by using NO donor treatment, co-culture of activated macrophages and mouse intestinal inflammation modeling, it is proved that ILC3 can bind NO produced by chemical reagents, cells and tissues in vivo, respectively. Further detection of the cytokine production capacity of ILC3 finds that NO can significantly inhibit the ability of ILC3 to produce pathogenic cytokine GM-CSF.

[0040] Further, the intestinal tissues of IBD patients are collected and analyzed for the NO binding level of ILC3, T cells and B cells, and it is found that the relative level of intracellular NO of ILC3 is negatively correlated with the activity of intestinal inflammation of patients. Therefore, the relative level of intracellular NO of intestinal ILC3 can be used to construct a detection reagent and kit for intestinal inflammatory diseases related to NO deficiency and the risk thereof.

[0041] The above two kits are combined to obtain the following kit with more comprehensive functions:

[0042] A kit for detecting inflammatory bowel disease caused by NO deficiency comprises quantitative reagents for detecting the expression amount of NO deficiency related proteins and quantitative reagents for detecting the intracellular NO content of intestinal immune cells.

[0043] The quantitative reagents for detecting the expression amount of NO deficiency related proteins comprise quantitative reagents for detecting NO synthase, quantitative reagents for detecting GM-CSF and quantitative reagents for detecting members of CYP450 capable of binding NO.

[0044] The intestinal immune cells are ILC3, T cells and B cells.

[0045] The third object of the present application is achieved by the following technical scheme:

[0046] An analysis system for detecting inflammatory bowel disease caused by NO deficiency, comprising:

[0047] A target expression detection device for detecting the expression of the NO deficiency related protein in a sample, which is the tissue or cells of a patient with colitis;

[0048] A target intracellular NO content detection device for detecting the intracellular NO content of each immune cell in a sample;

[0049] A drug analysis device for analyzing whether the patient is treated by supplementing the activity substance of the enzyme member capable of binding NO in CYP450 and the supplement amount and supplement method based on the detection results of the expression of the NO deficiency related protein and the intracellular NO content of each immune cell in a sample;

[0050] A result output device for outputting the results analyzed by the drug analysis device.

[0051] The analysis system can use the above-mentioned kit when in use.

[0052] The present application will be further described in detail below in combination with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 For human and mouse intestinal inflammation processes NOS2 Expression up-regulation. Figure 1 A is: the enzyme responsible for the synthesis of biologically active gas molecules (including: NO, H2S and CO, which are active and can participate in various physiological activities) in the human body (left) and the gene expression analysis of intestinal tissues of healthy controls (HC), Crohn's disease (CD) and ulcerative colitis (UC) patients in public databases (right). Figure 1 B is: a heat map showing Figure 1 The relative expression of the corresponding genes in the intestinal tissues of CD and UC patients in A compared with HC. Figure 1 C is: the gene expression in the intestinal tissues of each mouse intestinal inflammation model in the public database.

[0054] Figure 2 Nitric oxide synthesis-deficient mice showed aggravated anti-CD40-induced intestinal inflammation.

[0055] Figure 2 A is: a flowchart of mouse intestinal inflammation modeling analysis. Figure 2 B is: the body weight change of wild type and nitric oxide synthesis-deficient mice after intestinal inflammation modeling. Figure 2C. Colonic length of wild type and nitric oxide synthase deficient mice at day 3 and day 7 after dextran sodium sulfate-induced colitis. Figure 2 D. Histopathological analysis of colonic tissue of wild type and nitric oxide synthase deficient mice at day 3 and day 7 after dextran sodium sulfate-induced colitis. Figure 2 E. Neutrophil infiltration in colonic tissue of wild type and nitric oxide synthase deficient mice at day 3 and day 7 after dextran sodium sulfate-induced colitis. Figure 2 F. Secretion of GM-CSF, a pathogenic factor of LTi cells, in colonic tissue of wild type and nitric oxide synthase deficient mice at day 3 and day 7 after dextran sodium sulfate-induced colitis.

[0056] Values are expressed as mean ± S.E.M.; n = 5 mice / group. * indicates significant difference (p < 0.05) between nitric oxide synthase deficient mice and wild type mice. p <0.05).

[0057] Figure 3 To show that LTi cells can bind nitric oxide and are inhibited by nitric oxide.

[0058] Figure 3 A. Flow chart of co-culture of macrophages and LTi cells. Figure 3 B. Intracellular nitric oxide level of LTi cells co-cultured with macrophages. Figure 3 C. Flow chart of experiment of LTi cells treated with nitric oxide donor. Figure 3 D. Intracellular nitric oxide level of LTi cells treated with nitric oxide donor. Figure 3 E. LTi cells treated with nitric oxide donor at different concentrations, and cytokine secretion ability of the cells was detected. Figure 3 F. Flow chart of co-culture of macrophages and LTi cells. Figure 3 G. Cytokine secretion ability of LTi cells co-cultured with macrophages. Figure 3 H. C.r. Flow chart of infection experiment. Figure 3 I. Cytokine secretion ability of LTi cells in the intestine of mice after infection was detected.

[0059] Values are expressed as mean ± S.E.M.; n = 5 mice / group. * indicates significant difference (p < 0.05) between experimental group and control group. p <0.05).

[0060] Figure 4 To show the effect of cytochrome P450 deficiency on the ability of LTi cells in the intestine of mice to bind nitric oxide.

[0061] Figure 4 A. Flow chart of experiment of ILC3 cells of wild type and cytochrome P450 deficient mice treated with nitric oxide donor.Figure 4 B. Detection of NKp46 after treatment with a nitric oxide donor + ILC3 intracellular nitric oxide levels. Figure 4 C. Detection of ILC3 intracellular nitric oxide levels after treatment with a nitric oxide donor. Figure 4 D. Experimental scheme for ILC3 cell experiments with HET0016 treatment of wild type and cytochrome P450 deficient mice. Figure 4 E. Detection of NKp46 after treatment with HET0016 + ILC3 binding capacity for nitric oxide. Figure 4 F. Detection of ILC3 binding capacity for nitric oxide after treatment with HET0016.

[0062] Values are expressed as mean ± S.E.M.; n = 5 mice / group. * indicates significant difference between cytochrome deficient mice and wild type mice (p < 0.05). p <0.05).

[0063] Figure 5 Effect of cytochrome P450 deficiency on the ability of intestinal LTi cells to produce the pathogenic cytokine GM-CSF in mice.

[0064] Figure 5 A. Detection of cytokine production capacity of LTi cells from wild type and cytochrome P450 deficient mice. Figure 5 B. Experimental scheme for LTi cell experiments with HET0016 treatment of wild type and cytochrome P450 deficient mice. Figure 5 C. Detection of cytokine production capacity of LTi cells after treatment with HET0016.

[0065] Values are expressed as mean ± S.E.M.; n = 5 mice / group. * indicates significant difference between cytochrome deficient mice and wild type mice (p < 0.05). p <0.05).

[0066] Figure 6 Cytochrome inhibitor HET0016 alleviates anti-CD40 induced intestinal inflammation.

[0067] Figure 6 A. Scheme for the analysis of intestinal inflammation in mice. Figure 6 B. Body weight loss in wild type and nitric oxide synthase deficient mice 3 days after modeling. Figure 6 C. Colon length in wild type and nitric oxide synthase deficient mice 3 days after modeling. Figure 6 D. Histopathological analysis of colon tissue in wild type and nitric oxide synthase deficient mice 3 days after modeling. Figure 6E is: the neutrophil infiltration in the colon of wild type and nitric oxide synthesis deficient mice at day 3 after modeling. Figure 6 F is: the secretion of LTi cell pathogenic factor GM-CSF in the colon of wild type and nitric oxide synthesis deficient mice at day 3 after modeling.

[0068] The numerical values are expressed as mean ± S.E.M.; n = 5 mice / group. * indicates significant difference between HET0016 treated mice and control mice (p < 0.05). p <0.05).

[0069] Figure 7 To verify the above findings in human ILC3.

[0070] Figure 7 A is: human ILC3 cytochrome family member gene expression (blue) and protein sequence conservation (red) analysis (top). Diagram of secondary structure model of murine Cyp4f13 and human CYP4F3 (bottom). Figure 7 B is: detection of the ability of human blood T cells, ILC2 and ILC3 to bind nitric oxide. Figure 7 C is: experimental flow chart of HET0016 treatment of human ILC3. Figure 7 D is: detection of the ability of HET0016 treated human ILC3 to bind nitric oxide. Figure 7 E is: detection of the ability of HET0016 treated human ILC3 to secrete GM-CSF. Figure 7 F is: correlation analysis of ILC3 intracellular nitric oxide levels with Mayo scores, fecal calprotectin levels and blood C-reactive protein levels in UC patient tissues. DETAILED DESCRIPTION

[0071] The technical solutions of the present application are further illustrated below with experimental data.

[0072] The experimental methods used in the following examples and experimental examples are conventional methods unless otherwise specified.

[0073] The materials, reagents, etc. used in the following examples and experimental examples can be obtained from commercial channels unless otherwise specified.

[0074] The quantitative experiments in the following examples and experimental examples are all set up with three repeated experiments.

[0075] All mouse studies in the following examples and experimental examples were performed under the approval of the Ethics Committee of Peking University Health Science Center. All mice were housed and maintained in a specific pathogen-free facility with a 12-hour light / 12-hour dark cycle, ambient temperature of 20-24 °C, and humidity of 30-70%. Experiments were performed using 6-8-week-old and gender-matched mice. C57BL / 6J mice were purchased from the Department of Experimental Animal Science, Peking University Health Science Center. Nos2 - / - Mice were purchased from Jackson Laboratory, USA. Cyp4f13 - / - Mice were purchased from the Model Animal Research Center of Nanjing University.

[0076] Example 1

[0077] A medicine for treating inflammatory bowel disease, which is an oral medicine powder, with the content of HET0016 being not less than 2% as set by the results of mouse experiments, and the content of HET0016 in this example being 3%, other components including amino acids, fats, carbohydrates, minerals, vitamins, and trace elements.

[0078] Example 2

[0079] A medicine for treating inflammatory bowel disease, which is an oral medicine tablet, with the content of HET0016 being not less than 2% as set by the results of mouse experiments, and the content of HET0016 in this example being 5%, other components including cellulose and vitamins.

[0080] Example 3

[0081] A medicine for treating inflammatory bowel disease, which is an oral liquid, with the content of HET0016 in the solute being not less than 5% as set by the results of mouse experiments, and the content of HET0016 in the solute in this example being 50%, other components in the solute being amino acids.

[0082] Example 4

[0083] A medicine for treating inflammatory bowel disease, which is an oral liquid, the components of this example being HET0016 and water, and the solute being all HET0016.

[0084] Example 5

[0085] A medicine for treating inflammatory bowel disease, which is an enema medicine, with the content of HET0016 in the solute being not less than 2% as set by the results of mouse experiments, and the content of HET0016 in the solute in this example being 2.5%, other components in the solute including anti-inflammatory drugs, the anti-inflammatory drugs in this example being: mesalazine or hydrocortisone sodium succinate.

[0086] Example 6

[0087] A medicine for treating inflammatory bowel disease, the medicine is an injection medicine, the content of HET0016 in the solute is not less than 5% according to the results of mouse experiments, the content of HET0016 in the solute of the embodiment is 7%, and the other components of the solute include hypertonic glucose (or glucose and fat emulsion), electrolyte, vitamin, trace element, so as to achieve the purpose of nutritional support.

[0088] Example 7

[0089] A medicine for treating inflammatory bowel disease, the components include HET0016 and 5-aminosalicylic acid, wherein the HET0016 and 5-aminosalicylic acid are packaged separately, the 5-aminosalicylic acid is a tablet or a capsule, and the components in the HET0016 solution only include HET0016 and water.

[0090] Example 8

[0091] A medicine for treating inflammatory bowel disease, the components include HET0016 and mesalazine, wherein the HET0016 and 5 mesalazine are packaged separately, the mesalazine is a tablet or a capsule, and the components in the HET0016 solution only include HET0016 and water.

[0092] Example 9

[0093] A kit for detecting inflammatory bowel disease caused by nitric oxide deficiency, comprising quantitative reagents for detecting the expression amount of nitric oxide deficiency related proteins; the nitric oxide deficiency related proteins are a combination of iNOS, GM-CSF and CYP4F3. The quantitative reagents for detecting iNOS include primers of the gene thereof, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2; the quantitative reagents for detecting GM-CSF include primers of the gene thereof, the nucleotide sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4; and the quantitative reagents for detecting CYP4F3 include primers of the gene thereof, the nucleotide sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 6. NOS2 CSF2 CYP4F3

[0094] If the expression amount of iNOS is detected to be lower than a threshold value, it is indicated that the pathogenic cause of the inflammation patient may be nitric oxide deficiency, and further, if the expression amounts of CYP4F3 and GM-CSF are higher than a detection threshold value, it is indicated that the treatment measure of supplementing HET0016 may be effective, and once the expression amount of iNOS is higher than a detection threshold value, it is indicated that the treatment of supplementing HET0016 may be ineffective.

[0095] Example 10

[0096] ​​​A kit for detecting inflammatory bowel disease caused by nitric oxide deficiency, comprising quantitative reagents for detecting the content of intracellular nitric oxide of intestinal immune cells; the immune cells are ILC3, T cells and B cells.

[0097] The flow cytometry antibodies for detecting immune cells are shown in Table 1; the reagent for detecting the level of intracellular nitric oxide is DAF-FADA (Bi Yun Tian, S0019S).

[0098] After obtaining the colon tissue cell sample of the patient with intestinal inflammation, ILC3, T cells and B cells are distinguished by flow cytometry antibody staining. Then the content of nitric oxide in each cell is labeled with a nitric oxide probe and detected by flow cytometry. If the relative level of intracellular nitric oxide of ILC3 cells is lower than the threshold value, it indicates that the cause of inflammation may be nitric oxide deficiency.

[0099] Example 11

[0100] A kit for detecting inflammatory bowel disease caused by nitric oxide deficiency, comprising quantitative reagents for detecting the expression amount of nitric oxide deficiency related proteins and quantitative reagents for detecting the content of intracellular nitric oxide of intestinal immune cells; the nitric oxide deficiency related proteins are any one or a combination of multiple of iNOS, GM-CSF and CYP4F3.

[0101] The quantitative reagent for detecting iNOS includes primers of its gene NOS2 with nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2; the quantitative reagent for detecting GM-CSF includes primers of its gene CSF2 with nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4; the quantitative reagent for detecting CYP4F3 includes primers of its gene CYP4F3 with nucleotide sequences shown in SEQ ID NO. 5 and SEQ ID NO. 6;

[0102] The flow cytometry antibodies for detecting immune cells are shown in Table 1; the reagent for detecting the level of intracellular nitric oxide is DAF-FA DA (Bi Yun Tian, S0019S).

[0103] If the expression amount of iNOS is lower than the threshold value, the expression amount of GM-CSF and CYP4F3 is higher than the threshold value, and the relative level of intracellular nitric oxide of ILC3 cells is lower than the threshold value, it indicates that the treatment of supplementing HET0016 may be effective for inflammatory bowel disease.

[0104] Example 12

[0105] An analysis system for detecting inflammatory bowel disease caused by nitric oxide deficiency, which is used in cooperation with the kit of Example 11, the system comprises:

[0106] Target point expression detection device: used for detecting the expression of the nitric oxide deficiency related protein in a sample; the sample is a tissue or cell of a patient with colitis;

[0107] Target point intracellular nitric oxide content detection device: used for detecting the intracellular nitric oxide content of each immune cell in a sample;

[0108] Drug analysis device: based on the detection results of the expression of the nitric oxide deficiency related protein and the intracellular nitric oxide content of each immune cell in a sample, analyze whether the patient is treated by supplementing HET0016 and the supplement amount and supplement method of HET0016;

[0109] Result output device: used for outputting the results analyzed by the drug analysis device.

[0110] Experimental Example 1

[0111] During human and mouse intestinal inflammation NOS2 Expression up-regulation:

[0112] In this experiment, the enzyme genes responsible for synthesizing biologically active gas molecules (including NO, H2S and CO) in humans were first selected. These biologically active gas molecules are active and participate in various physiological processes, but it is still unclear whether they are involved in IBD. The colon tissue Bulk RNA-seq of UC patients ( Figure 1 A and B) and mouse intestinal inflammation models ( Figure 1 C) were downloaded from the GEO database, and the obtained data were analyzed by bioinformatics analysis method to determine the genes synthesizing nitric oxide NOS2 up-regulated during human and mouse intestinal inflammation. The results are shown in Figure 1 .

[0113] RNA-seq analysis of colon tissue of ulcerative colitis (Ulcerative Colitis, UC) patients in the GEO database showed that the expression of NOS2 gene synthesizing nitric oxide in UC patients was up-regulated compared with healthy controls ( Figure 1 A). Among various enzyme genes responsible for synthesizing gas molecules, NOS2 the expression change was the largest ( Figure 1 B). RNA-seq analysis of colon tissue of each mouse intestinal inflammation model in the GEO database, NOS2 was up-regulated in each mouse intestinal inflammation ( Figure 1 C).

[0114] The above results show that the NOS2 synthesizing nitric oxide is up-regulated during human and mouse intestinal inflammation.

[0115] Experimental Example 2

[0116] The effect of nitric oxide deficiency on anti-CD40-induced intestinal inflammatory response in mice:

[0117] This experiment used 6-8 week old, sex-matched wild-type C57BL / 6 mice and inducible nitric oxide synthase. Nos2 A mouse model was constructed using the deletion technique, divided into an anti-CD40-induced colitis phase (3 days) and a recovery phase (7 days). Mouse weight was recorded daily, and weight change curves were plotted. Mice were sacrificed on days 3 and 7, and colon samples were collected for length photography, H&E staining, and flow cytometry analysis. Results are as follows: Figure 2 As shown.

[0118] Anti-CD40-induced colitis models were established using wild-type mice and nitric oxide synthesis-deficient mice, and analyses were performed on days 3 and 7. Figure 2 A). Changes in body weight between the two groups of mice ( Figure 2 B) Colon length on days 3 and 7 of colitis ( Figure 2 C) Pathological changes in the colon ( Figure 2 D) Colonic neutrophil infiltration ( Figure 2 E) and the expression differences of colonic LTi cell effector factors GM-CSF and IL-22 ( Figure 2 F).

[0119] The above results indicate that mice with nitric oxide synthesis defects experience aggravated enteritis in an anti-CD40-induced enteritis model.

[0120] Experimental Example 3

[0121] LTi cells bind to and are inhibited by nitric oxide:

[0122] Sorted LTi cells were cultured in complete medium supplemented with 10 ng / ml recombinant mouse IL-7 (PeproTech), 10 ng / ml recombinant human IL-2 (PeproTech), and 10 ng / ml recombinant mouse SCF (PeproTech). Macrophages stimulated with 200 mg / ml LPS for 24 h were co-cultured for 24 h, or treated with 1 mM SNP or 100 μM NOC-18 for 3 h. Intracellular nitric oxide levels or GM-CSF production in LTi cells were then measured. Six- to eight-week-old, sex-matched wild-type C57BL / 6 mice and inducible nitric oxide synthase were used. Nos2 A rodent citrate infection model was established using mice with the deletion feature, and the production of GM-CSF in ILC3 was detected on day 10.

[0123] The results are as follows Figure 3 As shown. Activated macrophages were co-cultured with LTi cells as nitric oxide donors (Figure 3 A), the level of nitric oxide in LTi cells was increased compared with co-culture with non-activated macrophages (B). Figure 3 B). The cultured LTi cells were treated with nitric oxide donor (C). Figure 3 C), the level of nitric oxide in LTi cells was also increased (D). Figure 3 D). The cultured LTi cells were treated with gradient concentration of nitric oxide donor, the production of pathogenic cytokine GM-CSF was inhibited in a concentration-dependent manner (E). Figure 3 E). LPS-activated macrophages were co-cultured with LTi cells (F). Figure 3 F), the production of pathogenic cytokine GM-CSF in LTi was also inhibited (G). Figure 3 G). Wild type mice and nitric oxide synthase deficient mice were infected with Citrobacter rodentium and examined at day 10 (H). Figure 3 H), the production of pathogenic cytokine GM-CSF in colon LTi of nitric oxide synthase deficient mice was significantly higher than that of wild type mice (I). Figure 3 I).

[0124] The above results show that LTi cells can bind exogenous nitric oxide, and the secretion of cytokines is inhibited by the bound nitric oxide.

[0125] Experimental Example 4

[0126] Nitric oxide binds to LTi cells through cytochrome P450:

[0127] In this experiment, 6-8 week old, gender-matched wild type C57BL / 6 mice and cytochrome P450 knockout mice were sorted for LTi cells and cultured in complete medium supplemented with 10 ng / ml recombinant mouse IL-7 (PeproTech), 10 ng / ml recombinant human IL-2 (PeproTech), and 10 ng / ml recombinant mouse SCF (PeproTech). The level of nitric oxide in LTi cells was detected after 3h treatment with 1 mM SNP. Wild type LTi cells in culture were treated with 100 μM HET0016 for 12h, and then treated with 1 mM SNP for 3h, and the level of nitric oxide in LTi cells was detected. The results are shown in Figure 4

[0128] The ability of wild type mice and cytochrome P450 deficient mice to bind nitric oxide produced by chemical reagents was detected (A). Figure 4 A), the ability of cytochrome P450 deficient LTi cells to bind nitric oxide was reduced (B-C). Figure 4 B-C). The cultured LTi cells were treated with cytochrome inhibitor HET0016 (D). Figure 4 D), the ability of LTi cells to bind nitric oxide was further reduced (E). Figure 4 ​E-F).

[0129] The above results show that nitric oxide is bound on LTi cells by cytochrome P450.

[0130] Experimental Example 5

[0131] Cytochrome P450 promotes LTi cells to produce GM-CSF:

[0132] In this experiment, 6-8 week old, gender-matched wild-type C57BL / 6 mice and cytochrome P450 deficient mice were sorted for LTi cells and stimulated with 1 ng / ml IL-23 and 1 ng / ml IL-1β for 3 h, and the production of GM-CSF was detected. Wild-type LTi cells cultured in vitro were treated with 100 μM HET0016 for 6 h in advance, and finally stimulated with 1 ng / ml IL-23 and 1 ng / ml IL-1β for 3 h, and the production of GM-CSF was detected. The results are shown in Figure 5 .

[0133] As shown in Figure 5 , the cytokine production of intestinal LTi cells of wild-type mice and cytochrome P450 deficient mice was detected, and the ability of cytochrome P450 deficient LTi cells to produce GM-CSF was reduced Figure 5 (A). The cultured LTi cells were treated with cytochrome inhibitors HET0016 Figure 5 (B), and the ability of LTi cells to produce GM-CSF was reduced Figure 5 (C).

[0134] The above results show that the activity of cytochrome P450 promotes LTi cells to produce GM-CSF.

[0135] Experimental Example 6

[0136] HET0016 can alleviate intestinal inflammation caused by nitric oxide deficiency:

[0137] In this experiment, 6-8 week old, gender-matched inducible nitric oxide synthase Nos2 deficient mice were used to construct an anti-CD40 induced colitis model, and the mice were treated with or without 10 mk / kg / day of HET0016 at the same time. The body weight of the mice was recorded every day, and the body weight change curve was drawn. The mice were sacrificed on the 3rd day, and the colon was taken for length photography, H&E staining and flow cytometry analysis. The results are shown in Figure 6 .

[0138] Nitric oxide synthase deficient mice were used to construct an anti-CD40 induced colitis model, and were treated with or without HET0016 every day, and were detected on the 3rd day Figure 6 (A). The body weight change of the two groups of mice with colitis on the 3rd dayFigure 6 B) colon length ( Figure 6 C) colon pathology ( Figure 6 D) colon neutrophil infiltration ( Figure 6 E) and differential expression of colon LTi cell effector factors GM-CSF and IL-22 ( Figure 6 F).

[0139] The above results show that anti-CD40-induced intestinal inflammation is alleviated in the HET0016-treated group.

[0140] Experimental Example 7

[0141] Human ILC3 can also bind nitric oxide via cytochrome P450 and is inhibited by HET0016:

[0142] Bulk RNA-seq of human intestinal ILC3 was downloaded from GEO database to analyze the expression of each member of cytochrome P450 family in human ILC3 and the similarity of their protein amino acid sequences with mouse CYP4F13. Human blood samples were treated with 1 mM SNP for 3 h and the level of nitric oxide in ILC3 cells was detected before and after treatment. Human intestinal ILC3 cells were pretreated with 100 μΜ HET0016 and then treated with 1 mM SNP to detect their ability to bind nitric oxide or stimulated with 1 ng / ml IL-23 and 1 ng / ml IL-1β to detect their ability to produce GM-CSF. The relative level of nitric oxide in intestinal ILC3 cells of UC patients was detected and correlation analysis was performed with their disease activity. The results are shown in Figure 7 .

[0143] Human ILC3 expresses a protein CYP4F3 highly homologous to mouse CYP4F13 ( Figure 7 A). Human ILC3 can bind nitric oxide produced by chemical reagents ( Figure 7 B). Cytochrome inhibitor HET0016 treatment can inhibit the nitric oxide binding ability of human ILC3 ( Figure 7 C-D) and the GM-CSF production ability of human ILC3 ( Figure 7 E). The relative level of nitric oxide in intestinal ILC3 of UC patients is negatively correlated with the activity of intestinal inflammation ( Figure 7 F).

[0144] The above results show that human ILC3 also expresses a member of cytochrome P450 family that can bind nitric oxide, and HET0016 can inhibit its nitric oxide binding ability and GM-CSF production ability.

[0145] The above embodiments are only part of the embodiments of the present application and cannot cover the whole application. Based on the above embodiments and drawings, those skilled in the art can obtain more embodiments without creative effort, and the embodiments obtained without creative effort should be included in the protection scope of the present application.

Claims

1. Use of a member of CYP450 capable of binding NO as a target in developing or designing a product with the function of treating and / or preventing inflammatory bowel disease, wherein the member of CYP450 capable of binding NO is CYP4F3.

2. Use according to claim 1, characterized in that: The product is a drug or a detection reagent.

3. Use of a substance with inhibitory effect on the enzyme activity of a member of CYP450 capable of binding NO in preparing a drug for treating and / or preventing inflammatory bowel disease, wherein the member of CYP450 capable of binding NO is CYP4F3, and the substance with inhibitory effect on the enzyme activity of the member of CYP450 capable of binding NO is HET0016.

4. Use according to claim 3, characterized in that: The drug is an oral drug, an enema drug or an injection drug; the oral drug is a powder, a tablet or an oral liquid.

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